Crotoxin Administration for Cancer Treatment and Pain Relief

ABSTRACT

Disclosed is a method for treating cancer and/or pain using an intra-patient dose escalation procedure to deliver dosages of crotoxin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application No. 61/261,333, filed Nov. 14, 2009 and incorporatedherein by reference.

FIELD OF THE INVENTION

The invention relates to compositions and methods for the treatment ofcancer. More specifically, the invention relates to compositionscomprising snake venoms and methods for their safe administration forcancer treatment.

BACKGROUND OF THE INVENTION

In 2005, 7.6 million people worldwide died of cancer. Based onprojections, cancer deaths will continue to rise with an estimated 9million people dying from cancer in 2015, and 11.4 million dying in2030. Each year in the European Union (EU) countries, nearly two millionpeople are diagnosed with cancer and there are over one million deathsfrom the disease (Cancer Research UK). It is estimated that there arenearly three million people alive in the EU who have received adiagnosis of cancer in the last five years (Cancer Research UK).

There are over 50 drugs used in cancer treatment, but many of thesedrugs have an average efficacy of about 20% and produce significant sideeffects. Cancer patients often forego treatment in order to avoid thedecreased quality of life associated with side effects such as nausea,vomiting, loss of appetite, weight loss, generalized weakness, and hairloss. In fact, there is a significant demand for drugs that counteractthe side-effects of anti-cancer therapy (e.g., Procrit®).

Snake venoms have demonstrated cytotoxic activity on tumour cell linesin vitro, although their anticancer properties in animal models havebeen studied only in a limited manner. The venoms from cobras containlarge amount of basic, non-enzymatic peptides of molecular weight 6.8kDa, generically called Cardiotoxin, which display cytolytic activity ona broad variety of tumor cells, and to a lesser extent, normal cells.Crotoxin, isolated from the venom of the South American rattlesnake,Crotalus durissus terrificus, has proven to have significant and broadacting anti-tumour activity both in-vitro and in-vivo. Its increasedaffinity for cancer cells is due primarily to the target subunit (A,crotapotin) and cell death is brought about by the B subunit(Crotactine) through the membrane-disrupting enzymatic activity. It hassignificant potential as a therapeutic agent, but it also hassignificant neurotoxic activity. Crotoxin, however, induces tolerance toits neurotoxic effects without altering the cytolytic properties,permitting the use of doses above that which would normally be lethal tothe host. Mice injected daily with progressively increasing doses ofCrotoxin develop tolerance to the lethal action of the toxin. Treatedmice tolerated daily doses of Crotoxin 20 to 35 fold higher than theoriginal LD50, without the characteristic signs of toxicity. It has asignificantly higher lethal activity toward cancerous cells than normalcells, and this effect has been presumed to be due to the alteredmake-up of the cell membranes produced by rapidly proliferating cells.The sensitivity of cancer cells to Crotoxin has been associated withtheir expression of epidermal growth factor, a surface receptorassociated with malignancy. Cancer cell lines with the highestsensitivity to Crotoxin include lung, CNS, and melanoma.

Crotoxin has been used in several human safety studies alone and incombination with Cardiotoxin (a combination of Crotoxin and Cardiotoxinin 1:1 ratio is known as VRCTC310) in patients with refractory cancer.Crotoxin, administered by intramuscular injection, was well toleratedwhen administered alone or in combination with Cardiotoxin, with amaximum tolerable dose (MTD) of 0.21 mg/m². Upon intramuscular injectionCrotoxin achieves maximal circulating levels at 1 hour and iseffectively cleared within 24 hours. Several severely ill patients havebeen reported to respond to treatment with reduced tumor burden,amelioration of pain, and improved quality of life. Givenintramuscularly, Crotoxin gives objective clinical responses in dosesclose to the maximum tolerated dose (MTD). Preclinical animal datasuggests that, by using a dose escalation protocol, the administrationof very high doses can be achieved without adverse toxicity because thehost becomes tolerant to the neurotoxic effects.

To date, subjects treated with Crotoxin have an overall response rate of43% in a variety of tumour types. When compared to current biologictherapies, Crotoxin appears to be almost twice as active as its nearestcompetitor. While many other biologic therapies must be combined withconventional treatments and only serve to extend survival by marginalamounts, Crotoxin is a monotherapy, and has demonstrated sufficientactivity to results in complete remissions in a relatively highpercentage of terminally ill subjects under protocols that have not yetbeen optimized. It appears that Crotoxin has its highest cytocidalactivity against highly malignant cancers, yet the side-effect profileis quite mild in comparison to other forms of chemotherapy orradiotherapy. Side-effects (e.g., diplopia, strabismus, ptosis,peripheral blockade of neuromuscular transmission) do exist, however,and have been reported in dose tolerance studies (Cura, J. et al.,“Phase I and Pharmacokinetics Study of Crotoxin (Cytotoxic PLA2,NSC-624244) in Patients with Advanced Cancer,” Clinical Cancer Research,Vol. 8, April 2002, p. 1033-1041).

What would be of great benefit, would be the development of methods foroptimizing higher dosage levels of Crotoxin while minimizing theneurotoxic side-effects.

SUMMARY OF THE INVENTION

The invention relates to Crotoxin compositions for intravenousadministration and dosing regimens for administering those compositionsto provide a cytotoxic effect on cancer cells and tumors withoutassociated neurotoxic side-effects. Compositions of the inventioncomprise Crotoxin doses of from about 0.04 to about 0.32 mg/m² (about0.0012 to about 0.01 mg/Kg) suitable for intravenous administrationusing an intra-patient dose escalation procedure. The invention alsocomprises a method for treating cancer comprising administering to acancer patient a dosage of from about 0.04 to about 0.32 mg/m² (0.0012to 0.01 mg/Kg), the dosage being intravenously administered in anintra-patient dose escalation procedure, the intra-patient doseescalation procedure comprising administering sequentially about 0.04mg/m²/day for a period of 3-5 days, about 0.12 mg/m²/day for a period of3-5 days, about 0.16 mg/m²/day for a period of 3-5 days, about 0.20mg/m²/day for a period of 3-5 days, about 0.24 mg/m²/day for a period of3-5 days, about 0.28 mg/m²/day for a period of 3-5 days, and about 0.32mg/m²/day for a period of 3-5 days. In some aspects, the procedure mayfurther comprise a two-day non-treatment period between each 3- to 5-daytreatment period. In some aspects, the procedure may also compriseadministration by saline drip over a two-hour period.

The invention also relates to methods of use of Crotoxin comprisingintra-patient dose escalation of Crotoxin injections for the treatmentof pain and/or for the treatment of neurologic and/or neuromusculardisorders. Pain may be associated with cancer, with arthritis, withmultiple sclerosis, or any of a variety of sources of pain. In oneaspect, the intra-patient dose escalation protocol is effective for thealleviation of pain associated with pancreatic cancer.

The invention also relates to methods of use of Crotoxin injectionsprovided in an intra-patient dose escalation protocol in conjunctionwith the administration of aspirin (acetylsalicylic acid) for thealleviation of pain.

DETAILED DESCRIPTION

The inventors have developed an intra-patient dose escalation procedurefor intravenous administration of Crotoxin for the treatment of cancer.The method may also be used for administration of Crotoxin for thetreatment of pain and for the treatment of diseases associated withautoimmunity and inflammation. In the procedure, Crotoxin doses of fromabout 0.04 to about 0.32 mg/m² (0.0012 to 0.01 mg/Kg) are administereddaily by intravenous administration over a 2-hour period by saline drip.The definition of DLT (dose-limiting toxicity) in the context of thisprocedure is described as inability of a patient to tolerate doseescalation twice. Should Crotoxin-related toxicity occur, as evidencedby neurological side-effects resulting from peripheral blockade ofneuromuscular transmission, a patient is given the previous dose foranother cycle (3-5 days), and upon completion of the repeated cycle,2^(nd) dose escalation is attempted. If the patient responds withCrotoxin-related toxicity after the 2^(nd) dose escalation attempt, theescalation is abandoned for this patient and the dose to whichescalation was made is assessed as DLT for this patient. The patient isallowed to remain on the highest tolerated dose for another 4 weeks,subject to clinical assessment from the treating physician. After 4weeks, tumor assessment is performed to assess potential efficacy of thedosage. Example dosing regimens are shown in Tables 1 and 2.

Briefly the method for treating cancer comprises administering to acancer patient at least one Crotoxin dosage of from about 0.04 to about0.32 mg/m², the dosage being intravenously administered in anintra-patient dose escalation procedure, the intra-patient doseescalation procedure comprising consecutive administration of about 0.04mg/m²/day for a 3-5 day treatment period, about 0.12 mg/m²/day for a 3-5day treatment period, about 0.16 mg/m²/day for a 3-5 day treatmentperiod, about 0.20 mg/m²/day for a 3-5 day treatment period, about 0.24mg/m²/day for a 3-5 day treatment period, 0.28 mg/m²/day for a 3-5 daytreatment period, and 0.32 mg/m²/day for a 3-5 day treatment period,wherein each successive dose escalation is conditioned upon the absenceof dose-limiting toxicity from the previous dose. The intra-patient doseescalation procedure may include a two-day non-treatment period betweeneach treatment period. For example, treatment may be performed duringweekdays (i.e., Monday through Friday), with a break from treatment onthe weekend.

The dosage provided in the method may be, for example, administeredintravenously, such as by saline drip. In one aspect, administration maybe performed by intravenous saline drip over about a two hour period.

The invention also provides a method for treating pain in a patientcomprising administering to the patient at least one Crotoxin dosage offrom about 0.04 to about 0.32 mg/m², the dosage being intravenouslyadministered in an intra-patient dose escalation procedure, theintra-patient dose escalation procedure comprising consecutiveadministration of about 0.04 mg/m²/day for a 3-5 day treatment period,about 0.12 mg/m²/day for a 3-5 day treatment period, about 0.16mg/m²/day for a 3-5 day treatment period, about 0.20 mg/m²/day for a 3-5day treatment period, about 0.24 mg/m²/day for a 3-5 day treatmentperiod, 0.28 mg/m²/day for a 3-5 day treatment period, and 0.32mg/m²/day for a 3-5 day treatment period, wherein each successive doseescalation is conditioned upon the absence of dose-limiting toxicityfrom the previous dose. In various aspects, the pain may be associatedwith cancer, arthritis, multiple sclerosis, and/or other diseaseconditions which are associated with pain. In certain aspects, the painmay be associated with pancreatic cancer, which has been described to bea particularly painful condition, with pain at later stages of thedisease being refractory to many forms of pain relief.

TABLE 1 Dosing Regimen #1 Days (starting Monday), Dose CROTOXIN notreatment Duration Level Daily Dose on weekends In days 1 0.04 mg/m²/day1-5 5 2 0.08 mg/m²/day  8-12 5 3 0.12 mg/m²/day 15-19 5 4 0.16 mg/m²/day22-26 5 5 0.20 mg/m²/day 29-33 5 6 0.24 mg/m²/day 36-40 5 7 0.28mg/m²/day 43-47 5 8 0.32 mg/m²/day 50-54 5

TABLE 2 Dosing Regimen #2 Days (starting Monday), Dose CROTOXIN notreatment Duration Level Daily Dose on weekends In days 1 0.04 mg/m²/day1-3 3 2 0.08 mg/m²/day 4-5, 8-10 5 3 0.12 mg/m²/day 11-12, 15 3 4 0.16mg/m²/day 16-18 3 5 0.20 mg/m²/day 19, 22-24 4 6 0.24 mg/m²/day 25-26,29 3 7 0.28 mg/m²/day 30-32 3 8 0.32 mg/m²/day 33, 36-37 3

For the treatment of pain, an intra-patient dose escalation protocol maycomprise intravenous administration as provided in Table 1 and/or Table2, or may comprise Crotoxin injection with or without the administrationof aspirin, in a dosing regimen comprising a minimal dose of about 0.05cc or about 0.1 cc given for a period of 5 days, followed by incrementalincreases in dosage administered for similar periods of time (e.g., 3-5days). Diphenhydramine (e.g., Benadryl®) may also be pre-administered orco-administered to minimize a reaction to the Crotoxin.

The invention may be further described by means of the followingnon-limiting examples.

EXAMPLES Multi-Patient 2-Cohort Study

Individuals selected for the study are adult patients withhistologically confirmed advanced solid tumors who have progresseddespite standard therapy, or for whom no standard therapy exists; havean ambulatory PS (ECOG 0-1); have tumour evaluation made within 28 daysbefore study drug administration (patients with non-measurable lesionsaccording to the RECIST guidelines not previously irradiated are allowedto enter the trial); have completed radiotherapy or chemotherapy or anyother anticancer therapy (including experimental therapy) more than 4weeks prior to enrollment into the trial and must have recovered fromall acute side effects of these treatments; have a life expectancygreater than 3 months; have an age between 18 and 75 years; have normalmarrow function with normal haematological parameters (Hb≧10 g/dl,WBC≧4.0×10^(9/)L, neutrophil count≧2.0×10⁹/L and platelets≧100×10⁹/L);have no medically significant impairment of cardiac or respiratoryfunctions; have adequate hepatic function with total bilirubin ≦1.5×Nand transaminases ≦2.5×N (≦5×N in case of liver metastasis); have nohistory of prior severe allergic reactions to venoms; have creatinineclearance ≦50 mL/min.

Additionally, patients must already be on stable doses of any drugswhich may affect hepatic drug metabolism or renal drug excretion (e.g.,non-steroidal anti-inflammatory drugs, barbiturates, narcoticanalgesics, probenecid), they should not be pregnant or planning tobecome pregnant, should not be known to have brain metastases orleptomeningeal involvement (CT-scan or MRI is not required to rule thisout unless there is clinical suspicion of central nervous systeminvolvement), should not have pleural effusion/ascites, cystic lesionsor bone metastases, should not be receiving any other experimental oranti-cancer therapy within 30 days before first study drugadministration (except antalgic radiotherapy and hormonotherapy), shouldnot have a history of other malignancies (except for patients with acancer free interval of greater than or equal to 5 years after treatmentcompletion or patients with prior history of adequately treated basalcell carcinoma of the skin or carcinoma in situ of the cervix), shouldnot have had recent major surgery (within 21 days) should not have arecent history of weight loss greater than 10% of current body weight,should not have serious intermittent medical illnesses which wouldinterfere with the ability of the patient to carry out the treatmentprogram, should not be on chronic steroid medication (greater than 20mg/day), and should not have primary or paraneoplastic myastheniagravis.

Crotoxin doses of about 0.04 to 0.32 mg/m² (0.0012 to 0.01 mg/Kg) areadministered using an intra-patient dose escalation procedure. Thedefinition of DLT (dose-limiting toxicity) in the context of this studyis described as inability of dose escalation twice. ShouldCrotoxin-related toxicity occur, the patient is given the previous dosefor another cycle (5 days) upon which 2^(nd) dose escalation isattempted. If the patient responds with drug treatment relatedCrotoxin-related toxicity after the 2^(nd) dose escalation attempt, theescalation is abandoned for this patient and the dose to whichescalation was made is assessed as DLT for this patient. The patient isoffered to remain on the highest tolerated dose for another 4 weeks,subject to clinical assessment from the treating physician. After 4weeks, tumor assessment is performed to assess potential efficacy of thetested compound.

Determination of general MTD may be done as follows: first, a patient istreated up to a 0.32 mg/m² or a lower tolerated dose. This dose iscalled the i^(th) target ceiling dose (TCD_(i)). Next, two patients willbe treated up to the TCD_(i). If no Crotoxin-related toxicity(dose-limiting toxicity, DLT) is encountered at the TCD_(i), then theTCD_(i) is the MTD. If two or more DLT are encountered, then 3 newpatients are included to receive the next lower dose (TCD_(i−1)), etc.If one DLT is encountered, then 3 more patients are added to receivedosages up to the TCD_(i). If no DLT is encountered among those 3 newpatients, then the TCD_(i) is MTD. If one or more DLT is encounteredamong those 3 new patients, then 3 new patients are included up toTCD_(i−1), etc.

Crotoxin is administered daily by intravenous administration over a2-hour period by saline drip. Each patient will receive Ranitidine 50 mg(antiemetic) and Polaramine 10 mg I.V. 10 mg (antihistamine)intravenously prior to treatment to minimize the potential foranaphylaxis.

Patients are treated as out-patients, attending the clinic daily fortreatment, monitored at the clinic for the duration of the infusion (2hours), and observed for 30 minutes following the infusion of the drug.If adverse events are to occur (anaphylaxis or neurotoxicity), they areexpected to manifest themselves during the administration of the drugallowing interruption of treatment (stopping of the infusion) ascirculating levels of the drug will rapidly fall once administration hasceased. The procedure to suppress the potential for anaphylaxis isfollowed in a standard protocol employed for any agent that can inducehypersensitivity reaction (e.g., such procedures are employed whenadministering Radio Contrast Media and monoclonal antibody therapiessuch as trastuzumab or cetuximab).

Crotoxin Administration Alleviates Pain Related to Pancreatic Cancer andMultiple Sclerosis

A female patient with both pancreatic cancer and multiple sclerosisexperienced such pain that it was not alleviated by medications such ashydromorphone and/or fentanyl. She began a dose escalation protocolcomprising injectable Crotoxin starting at 0.01 cc. A dose of 0.1 cccontains 40 micrograms Crotoxin or about 0.6 micrograms/kilogram. Shehas been maintained at the 0.1 cc dose and reports being pain free andable to move muscles and joints without experiencing pain. Of even moresignificance, she reports experiencing no pancreatic pain. The decreasein pain was of such magnitude that the patient experienced it as a senseof euphoria.

1. A method for treating cancer comprising administering to a cancerpatient at least one Crotoxin dosage of from about 0.04 to about 0.32mg/m², the dosage being intravenously administered in an intra-patientdose escalation procedure, the intra-patient dose escalation procedurecomprising consecutive administration of about 0.04 mg/m²/day for a 3-5day treatment period, about 0.12 mg/m²/day for a 3-5 day treatmentperiod, about 0.16 mg/m²/day for a 3-5 day treatment period, about 0.20mg/m²/day for a 3-5 day treatment period, about 0.24 mg/m²/day for a 3-5day treatment period, 0.28 mg/m²/day for a 3-5 day treatment period, and0.32 mg/m²/day for a 3-5 day treatment period, wherein each successivedose escalation is conditioned upon the absence of dose-limitingtoxicity from the previous dose.
 2. The method of claim 1 wherein theintra-patient dose escalation procedure further comprises a two-daynon-treatment period between each treatment period.
 3. The method ofclaim 1 wherein the dosage is intravenously administered by saline drip.4. A method for treating pain in a patient comprising administering tothe patient at least one Crotoxin dosage of from about 0.04 to about0.32 mg/m², the dosage being intravenously administered in anintra-patient dose escalation procedure, the intra-patient doseescalation procedure comprising consecutive administration of about 0.04mg/m²/day for a 3-5 day treatment period, about 0.12 mg/m²/day for a 3-5day treatment period, about 0.16 mg/m²/day for a 3-5 day treatmentperiod, about 0.20 mg/m²/day for a 3-5 day treatment period, about 0.24mg/m²/day for a 3-5 day treatment period, 0.28 mg/m²/day for a 3-5 daytreatment period, and 0.32 mg/m²/day for a 3-5 day treatment period,wherein each successive dose escalation is conditioned upon the absenceof dose-limiting toxicity from the previous dose.
 5. The method of claim4 wherein the pain is associated with cancer.
 6. The method of claim 5wherein the cancer is pancreatic cancer.
 7. The method of claim 4wherein the pain is associated with arthritis.